DESCRIPTION: (Applicant's Abstract) Agonist stimulation of opioid receptors elicits an excitatory response manifest as an increase in intracellular free Ca2+ concentration ([Ca2+)i) that results from release of Ca2+ from intracellular stores in undifferentiated NG108-15 cells. The overall objective of this proposal is to employ electrophysiologic, pharmacologic and microfluorimetric techniques to characterize the pathway which mediates this opioid-induced excitation and determine how chronic exposure to opiates affects the response. Our first aim is to determine the link that couples opioid receptors to Ca2+ mobilization. We hypothesize that opioid-induced dissociation of beta gamma subunits from heterotrimeric inhibitory G proteins activates phospholipase C to mobilize Ca2+ from intracellular stores. This hypothesis provides a rational explanation for how delta receptors can activate effectors not traditionally thought to couple to inhibitory G proteins. Our second aim is to determine the pathway that links opioid receptors to the opening of Ca2+ channels in differentiated cells. In differentiated NG108-15 cells opioids evoke Ca2+ influx via an indirect pathway. We hypothesize that in differentiated cells, opioid induced activation of protein kinases leads to depolarization and subsequent recruitment of voltage-gated Ca2+ channels. This hypothesis may explain some effects of opioids on neuronal excitability. Our third aim is to characterize the effects of prolonged exposure to opioids on agonist-induced Ca2+ release. We hypothesize that chronic treatment of NG108-15 cells with opioids will desensitize opioid-induced [Ca2+]i increases. These studies are predicted to determine whether desensitization of opioid receptors affects differentially the various second messenger systems modulated by opioids. Our fourth aim is to determine the extent to which opioid-induced excitatory effects can be seen in primary tissue. We will test rat central and peripheral neurons grown in primary culture for opioid-induced increases in [Ca2+]i. Including an additional signaling pathway, possibly the phosphoinositide cascade, among the multiple second messenger systems modulated by opioids may be key to understanding the molecular mechanisms of drug abuse. Characterization of the excitatory responses elicited by opioids in NG108-15 cells will provide the basis for studies on cross talk between second messenger systems and how these relationships change during development and chronic stimulation.